Synthesis of steroids



United States Patent 4 Claims ABSTRACT OF THE DISCLOSURE 7B-hydroxyestranes are prepared by a two-step process. In the first step a 19-nor-A -androstene is subjected to the action of enzymes of a 7B-hydroxylating microorganism to form the corresponding 7B-hydroxy-l9-nor- A 'androstene derivative. The latter compound is then subjected to the action of enzymes of a 1dehydrogenating microorganism to yield the final 7fl-hydroxyestrane product.

This application is a division of application, Ser. No. 527,501, filed Feb. 15, 1966.

This invention relates to and has for its object the provision of an improved process for preparing 7B-hydroxyestranes and to the new steroids formed thereby.

It has been found that a 19-nor-A -androstene may be converted to a 7fi-hydroxyestrane derivative in high yield by a two-step process without any substantial formation of undesired by-products. In essence, therefore, the process of this invention entails subjecting a l9-nor-A -androstene to the action of enzymes of a 7fi-hydroxylating microorganism, whereby a corresponding 7B-hydroxy-19- nor- -androstene derivative is formed; and subjecting the latter to the action of enzymes of a l-dehydrogenating microorganism, to yield the desired 7fi-hydroxyestrane final product.

Among the suitable starting steroids are included any of the 19-nor-A -androstenes. The preferred starting steroids, however, are the 3,17-dioxygenated-19-nor-A androstenes, such as 19-nor-A -androstene-3,l7-dione, 19-nortestosterone, 19-nor-17ot-methyltestosterone and l9-nor-17u-ethynyltestosteroue.

In the first step of the process of this invention, the steroid substrate is subjected to the action of enzymes of a 7,6-hydroxylating microorganism, the reaction being carried out in the usual manner by culturing the microorganism in the presence of the steroid, or by treating the steroid with nonproliferating cells of the microorganism, or by intermixing the steroid with 7B-hydroxylating enzymes previously obtained from the microorganism. The conditions for such microbial reaction are well known in the art and are similar to those specified in US. Patent 3,179,698.

Any 7B-hydroxylating microorganism can be used as the source of the 7/3-hydroxylating enzyme. Such microorganisms include, inter alia, Diplodia natalensis, Aspergillus niger, T lzamnz'dium elegans, Coniothyrium hellebori and Phycomyces blakesleeanlls.

The process results in the preparation of the 7fi-hydroxy-19-nor- -androstene intermediates which are new compounds of this invention. The preferred intermediates are the 75-hydroxy-3,17-dioxygenated-19-nor-A -androstenes, such as 19-nor-A -androstene-7Bo1-3,17-dione, 7B-hydroxy 19 nortestosterone, 75 hydroxy-19-nor- 17a-methyltestosterone, and 7;8-hydroxy 19 nor 17aethylnyltestosterone.

These 718-hydroxy-19-nor-A -androstenes are then sub- 3,429,778 Patented Feb. 25, 1969 jected to the action of enzymes of l-dehydrogenating microorganisms, to yield the desired 7B-hydroxyestrane final products, the reaction being carried out in the usual manner by culturing the microorganism in the presence of the steroid, or by treating the steroid with non-proliferating cells of the microorganism, or by intermixing the steroid with l-dehydrogenating enzymes previously obtained from the microorganisms. Optimally the dehydrogenation is conducted with cel1-free extracts of l-dehydrogenating microorganisms, as by the method and with the enzymes described in US. Patent No. 3,047,469.

The second step of the process results in the formation of the final products, namely the 7fi-hydroxyestrane derivatives, which include the 7f3-hydroxy-3,l7-dioxygenated estranes, such as 7fl-hydroxyestrone, 7,8-hydroxyestradiol, 7fi-hydroxy 17oz methylestradiol and 7B-hydroxy-17aethynylestradiol.

The following examples illustrate the invention (all temperatures being in centigrade):

Example 1.7fi-hydroxy-l9-nor-A -androstene-3,17-dione A. Fermentation-Surface growth from each of three 10-day old agar slant cultures of Diplodia! natalensis (ATCC 9055), the slant containing as a nutrient medium (A):

Grams Glucose 10 Yeast extract 2.5 K HPQ, 1 Agar 20 Distilled water to 1 liter.

is suspended in 5 ml. of a 0.01% sodium lauryl sulfate aqueous solution. One ml. portions of the suspension are used to inoculate ten 250 ml. conical flasks, each containing 50 ml. of the following sterilized medium (E):

Distilled water to 1 liter.

After 72 hours of incubation at 25 with continuous rotary agitation (280 cycles per minute; 2 inch stroke), 10% (vol/vol.) transfers are made to forty 250 ml. conical flasks each containing 50 ml. of freshly sterilized medium B plus 500 micrograms/ml. of l9-nor-A -androstene-3,17-dione. The steroid is added by supplementing each flask 'with 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide containing mg./ml. of steroid. A total of 1.0 gram is used. After 7 days of further incubation, the contents of the flasks are pooled through a Seitz clarifying pad. The flasks, mycelium and pads are washed with successive 50 ml. portions of warm water. The combined filtrate and washings have a volume of 2,000 ml.

B. Isolation and Characterization.-The combined filtrate and washings (2,000 ml.) are extracted three times with 500 ml. portions 0 methyl isobutyl ketone. The combined methyl isobutyl ketone extracts are washed with water, dried over anhydrous sodium sulfate and concentrated to dryness under vacuum, leaving about 500 mg. of crude product. This material is chromatographed on a thin layer of silica Gel GF (Merck) with chloroform containing 5% (by volume) methanol as the developing solvent. The UV-absorbing band which moves with approximately 3/ 10 mobility of the substrate, 19-norandrostenedione, is eluated with a 1:1 (by volume) mixture of methanol and chloroform. After evaporating 01f the solvent, the residue is partitioned between chloroform and a 1:1 (by volume) mixture of water and methanol. The chloroform phase, upon evaporation under vacuum to dryness, yields crystalline 7fl-hydroxy-19-nor-A -androstene-3,l7dione. It is recrystallized from acetonehexane to yield about 15 ml. of the pure product, M.P. about 208-209 C.; [a] -|127 (C, 0.52, chloroform);

A313 241 m (e=15,800). 2?; 3370, 1730, 1639, 1603 cm. Example 2 Following the procedure of Example 1, but substituting an equivalent amount of 19-nortestosterone for the 19- nor-M-androstene 3,17 dione, 7,8 hydroxy-19-nor-A androstene-3,17-dione having a M.P. about 208-209 C. is obtained.

Example 3 Steroid substrate: Product 19-nor-17a-methyl- 7fi-hydroxy-19-nor-17atestosterone methyltestosterone 19-nor-17u-ethynyl- 7 ,B-hydroxy-19-nor-17 OL- testosterone ethynyltestosterone Example 4.7;8-hydroxyestrone by growing culture of Corynebacterium simplex A. Fermentation.Surface growth from a two-week old agar slant of Corynebacterium simplex (ATCC 6946), the slants containing as a nutrient medium (A):

Grams Glucose Yeast extract 2.5 K HPO 1 Agar 20 Distilled water to 1 liter.

is suspended in 5 m1. of 0.01% aqueous sodium lauryl sulfate solution. One ml. portions of this suspension are used to inoculate our 250 ml. Erlenmeyer flasks, each containing 50 ml. of the ollowing sterilized medium (B):

Grams Beef extract 1.5 Yeast extract 3 Peptone 6 Dextrose 1 Distilled water to 1 liter.

After 24 hours of incubation at 25 with continuous rotary agitation (285 cycles/minute; 2 inch stroke), 5% (vol/vol.) transfers are made to eight 250 ml. Erlenmeyer flasks each containing 50 ml. of freshly sterilized medium B. After 24 hours of further incubation, using the same conditions as described above, the steroid, (250 micrograms/ml.) is added by supplementing each flask with 0.25 ml. of a sterile solution (50 nag/ml.) of 7B-hydroxy- 19-nor-A -androstene-3,17-di0ne in N,N dimethylformamide. A total of 100 mg. is fermented. After 48 hours of further incubation, using identical conditions as described above, the contents of the flasks are pooled, and the broth is extracted three times with 200 ml. portions of methyl isobutyl ketone. Upon evaporation of the combined extract under vacuum to dryness, crystalline 7,9-

hydroxyestrone is obtained. It is recrystallized twice from acetone-hexane to yield about mg. of the pure product, M.P. 261-262", [aJ -i-ISO (dioxane),

75323490, 3280, 1720, 1621, 1587, 1503 cur Example 5 .7/3-hydroxyestrone by washed cells of Corynebacterium simplex Following the procedure of Example 4 with the exception that testosterone is used in place of 7,8-hydroXy-19- norandrostenedione, the cells of the culture of Corynebacterium simplex are harvested at the end of 72 hours by centrifugation. The packed cells are washed three times with a phosphate buffer containing 0.005 mole each of KH PO and N a H P O per liter and adjusted to pH 7.0. The washed cells are then suspended in the same phosphate bufler to a volume equal to one-quarter of the volume of the original culture. The substrate, 7fi-hydroxy- 19-norandrostenedione and the hydrogen acceptor, e.g., Z-methyl-naphthoquinone are added as their solutions in ethanol to give final concentrations of g/ ml. and 0.4 mM., respectively, the quantity of ethanol introduced being held within 5% of the total. The reaction mixture is allowed to stand at 30 for 4 to 6 hours, after which it is extracted twice with one-quarter of its volume of methyl isobutyl ketone. The methyl isobutyl ketone extract is washed twice with water and dried over anhydrous sodium sulfate. Upon evaporating off the solvent to dryness, 7/3- hydroxyestrone is obtained as crystalline residue. It is recrystallized from acetone-n-hexane, M.P. 261262, [a] (dioxane).

Example 6.7 3-hydroxyestrone by cell-free enzyme preparation from Coryizebacterium simplex Following the procedure of Example 5, the packed cells are placed in a mortar along with an equal amount by weight of alumina (finely powdered) and treated in a Raytheon magnetostrictive oscillator for 20 minutes. The sonicated mixture is centrifuged for ten minutes at 2000 G to remove cell debris and alumina. The substrate, 7,8- hydroxy-19-norandrostenedione (1 mg.) and the hydrogen acceptor, e.g., Z-rnethyl-naphthoquinone (1 mg.) are added to 2 ml. of this cell-free ring-A dehydrogenase preparation which has been diluted to 5 ml. with pH 7.0 phosphate buffer in the same manner as described in Example 5. The mixture is allowed to stand for one hour at 30 C. after which it is twice extracted with 1 ml. of methyl isobutyl ketone. The combined extract is chromatographed on paper using ethylene glycol as the stationary phase and a mixture of equal volumes of benzene and chloroform as the mobile phase. A spot moving with the same R (0.12) and exhibiting the same characteris tic color reactions as the 7/3-hydroxyestrone obtained in Example 4 is observed.

Similarly, by substituting the following I-dehydrogenating microorganisms for the Corynebacterium simplex in Examples 4, 5, and 6, the same products are formed: Nocardia restrictus ATCC14887, Pseudomonas testosteroni ATCC11996, Cylindrocarpon radicicola ATCC- llOll, and Mycobaclerium rhodochrous ATCC-4277.

The invention may be variously other-wise embodied within the scope of the appended claims.

What is claimed is:

1. A process for preparing a 7,9-hydroxy-A estratriene, which comprises subjecting a 19 non A androstene serially to the action of enzymes of the 7,3- hydroxylating microorganism Diplodia natalensis and enzymes of a l-dehydrogenating microorganism.

2. The process of claim 1 wherein the androstene is 19-nor-A -androstene-3,17-dione.

3. A process for preparing a 7fl-hydroxy-19-nor-A androstene, which comprises subjecting a l9-norA -androstene to the action of enzymes of Diplodia natalensis and recovering the 7,8 hydroxy 19 nor A androstene produced.

References Cited UNITED STATES PATENTS 7/1958 Wettstein et a1. 195-51 9/1959 Weintraub et a1. 195-51 Herzog et a1. 195-51 Thoma et a1. 195-51 Holmlund et a1. 19551 Edwards et a1. 19551 Pan et a1. 195-51 ALVIN E. TANENHOLTZ, Primary Examiner.

K46ba UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION patent 3, 429, 778 Dated February 25, 1969 Inventor) Samuel Cheng Pan et al.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

'- Column 2, line 41, change NH H PO to NH H PO line 62, change 0 to of.

Column 3, line 14, change 0 to of; line 22, change 9 to 19; line 53, change ollowing to following; line 62, change 285 to 280.

Column 4, line 65, claim 1, change non to nor.

SIGNED AN'D SEALED NOV 1 1959 (SEAL) Attest:

Edward M. Fletcher, Ir. WILLIAM saauym, m

mmissi Attesting Officer atents 

